Cardiovascular disease has become one of the most globally prevalent diseases, and autologous or vascular graft transplantation has been the main treatment for the end stage of the disease. However, there are no commercialized small-diameter vascular graft (SDVG) products available. The design of SDVGs is promising in the future, and SDVG preparation using an in vitro bioreactor is a favorable method, but it faces the problem of long-term culture of >8 weeks. Herein, we used different oxygen (O2) concentrations and mechanical stimulation to induce greater secretion of extracellular matrix (ECM) from cells in vitro to rapidly prepare SDVGs. Culturing with 2% O2 significantly increased the production of the ECM components and growth factors of human dermal fibroblasts (hDFs). To accelerate the formation of ECM, hDFs were seeded on a polycaprolactone (PCL) scaffold and cultured in a flow culture bioreactor with 2% O2 for only 3 weeks. After orthotopic transplantation in rat abdominal aorta, the cultured SDVGs (PCL–decellularized ECM) showed excellent endothelialization and smooth muscle regeneration. The vascular grafts cultured with hypoxia and mechanical stimulation could accelerate the reconstruction speed and obtain an improved therapeutic effect and thereby provide a new research direction for improving the production and supply of SDVGs.